Short Report: Influence of Centers for Disease Control Light Trap Position, Relative to a Human-Baited Bed Net, on Catches of Anopheles Gambiae and Culex Quinquefasciatus in Tanzania.

The best position for Centers for Disease Control (Atlanta, GA) light traps, in relation to human-occupied bed nets for trapping of host-seeking Anopheles gambiae Giles and Culex quinquefasciatus Say mosquitoes, was determined in Tanzania. Significantly higher catches were recorded for both species when the trap was positioned at the foot end of the bed, near the top of the net. Parity rates were significantly higher near the top of the net than at the level of the host. Since trap position affects the catch size and the proportion of infectious mosquitoes therein, standardized use of this sampling technique for estimating entomologic inoculation rates (i.e., the number of potentially infectious bites received over a certain period of time) is recommended

Human skin emanations in the host-seeking behaviour of the malaria mosquito Anopheles gambiae

Malaria is an infectious disease caused by a parasite ( Plasmodium spp.) that is transmitted between human individuals by mosquitoes, belonging to the order of insects, Diptera, family of Culicidae (mosquitoes) and genus of Anopheles (malaria mosquitoes). Mosquitoes feed on humans (and other animals) because they need blood for their reproduction. Like most other haematophagous insects, only the female mosquitoes bite and use the protein-rich blood meal for egg development. Whilst feeding on a person infected with malaria, the mosquito can be pick up the parasites from the blood stream. After a developmental period in the mosquito, the parasites can be transmitted to another person when the mosquito takes a next blood meal. Thus, malaria transmission depends largely on the characteristics of the mosquito population. Knowledge about the ecology, behaviour, infection level and size of the mosquito population is essential for the development, implementation and evaluation of control programs. Development of an adequate trapping device for monitoring the mosquito population is of high priority for ecological and epidemiological studies.Malaria is one of the most important human parasitic infectious diseases and one third of the world population is under threat of the disease. Most victims are found in the sub-Saharan countries of Africa. The Afrotropical malaria mosquito, Anopheles gambiae sensu stricto, is the most important vector since it strongly prefers to feed on humans. Like most anopheline species, An. gambiae s.s. is nocturnal and its host-seeking behaviour is mainly odour-mediated. Consequently, odour-baited traps are considered as possible monitoring devices. However, despite the important role of this mosquito in malaria transmission, knowledge regarding host odour components (or kairomones) that bring about the attraction to humans is limited. For the development of odour-baited traps, attractive host odours need to be identified. In this thesis a behavioural ecological investigation to the source, identification and production of kairomones for An. gambiae (henceforth simply termed 'malaria mosquitoes') is described.Source of kairomones for malaria mosquitoesSince the beginning of the century it has been recognised that malaria mosquitoes utilise host odours in their host-seeking behaviour. The source of these olfactory stimuli is expired air, the skin or both. Carbon dioxide present in expired air is an important kairomone for many haematophagous insects. For this reason carbon dioxide is often used in odour-baited traps. From field research (Chapter 2) we learnt that malaria mosquitoes can find their host in the absence of breath, and, thus, the presence of carbon dioxide is not compulsory for finding a host. This suggests that volatiles from the skin of the host also play a role in the attraction of malaria mosquitoes. The addition of skin volatiles to a carbon dioxide baited trap will probably bring about higher trap catches. Moreover, for logistic reasons, an odour-baited trap without presence of carbon dioxide is preferable. Carbon dioxide is highly volatile and can be delivered only by gas cylinders or dry ice (= frozen carbon dioxide), which is impractical in the African field situation. The composition of body odour is complex: more than 300 components have been identified. However, a synthetic blend of the complete human odour has not yet been synthesised. For this reason, the identification of some important components that attract malaria mosquitoes was initiated. A prerequisite for the identification was the entrapment of natural skin emanations separate from the skin. Sweat appeared to be an attractive complex olfactory stimulus since it is not artificial but rather true to nature (Chapter 4 and 5) in the bioassays in the laboratory and it forms the 'heart' of the thesis.The identification of kairomones for malaria mosquitoesSweat was collected from the foreheads of a number of volunteers, who performed exercises on a hometrainer in a warm and humid room. The behavioural response of the malaria mosquitoes to this fresh sweat was rather variable; they were attracted to some fresh sweat samples (Chapter 8 and 9) but not to others (Chapter 5 and 7). However, the response of the mosquitoes to sweat that had been incubated for two days at body temperature was stable, and all incubated sweat samples were attractive to the mosquitoes. It appeared that the incubation released volatile components that were attractive to mosquitoes. Sweat is basically a watery solution of lactic acid, urea and ammonia. After incubation the lactic acid and urea concentration had decreased and the ammonia concentration showed a distinct increase (Chapter 8 and 9). For this reason ammonia was tested in the bioassay. For the first time, malaria mosquitoes were attracted to a single component other than carbon dioxide, namely ammonia. Lactic acid is an essential kairomone for another mosquito species, the yellow fever mosquito Aedes aegypti . However, the selective removal of lactic acid from the sweat did not affect the reaction of malaria mosquitoes. Therefore, we conclude that lactic acid is not an essential component of attractive odour blends for malaria mosquitoes. Urea was not tested, as it is not volatile. The fact that attraction was sometimes found to the fresh sweat with a rather low concentration of ammonia indicates that components other than ammonia also play a role in the host-seeking behaviour of malaria mosquitoes. The identity of these components needs further exploration.The production of kairomones for malaria mosquitoesThe skin of humans (and other animals) forms a good habitat for some microorganisms (bacteria and fungi), together called the skin microflora. During the collection of sweat samples, microorganisms are taken up with the sweat. An exponential growth of microorganisms in the sweat samples is found during incubation (Chapter 4, 6 and 7). Sweat constituents are broken down into more volatile components by the growing microorganismal population and this appears to bring about the enhancement of the attractiveness of sweat to malaria mosquitoes (Chapter 9). Such processes also probably play a role in the production of kairomones on the skin. However, this needs further exploration.ConclusionsKairomones for malaria mosquitoes originate from the human skin, in addition to carbon dioxide from exhaled air. Microorganisms of the skin flora play an important role in the production of kairomones for malaria mosquitoes An. gambiae s.s.. Ammonia is one of the components responsible for the attraction of malaria mosquitoes to sweat.</p

Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems

Translational control of UIS4 protein of the host-parasite interface is mediated by the RNA binding protein Puf2 in Plasmodium berghei sporozoites

Copyright: © 2016 Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.UIS4 is a key protein component of the host-parasite interface in the liver stage of the rodent malaria parasite Plasmodium berghei and required for parasite survival after invasion. In the infectious sporozoite, UIS4 protein has variably been shown to be translated but also been reported to be translationally repressed. Here we show that uis4 mRNA translation is regulated by the P. berghei RNA binding protein Pumilio-2 (PbPuf2 or Puf2 from here on forward) in infectious salivary gland sporozoites in the mosquito vector. Using RNA immunoprecipitation we show that uis4 mRNA is bound by Puf2 in salivary gland sporozoites. In the absence of Puf2, uis4 mRNA translation is de-regulated and UIS4 protein expression upregulated in salivary gland sporozoites. Here, using RNA immunoprecipitation, we reveal the first Puf2-regulated mRNA in this parasite.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) grants PTDC/SAU-MIC/122082/2010 and PTDC/BIA-BCM/105610/2008 to GRM, and SFRH/BPD/72619/2010 to PAGCS.info:eu-repo/semantics/publishedVersio

The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

&lt;b&gt;Background&lt;/b&gt; Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. &lt;b&gt;Methods:&lt;/b&gt; The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. &lt;b&gt;Results:&lt;/b&gt; The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. &lt;b&gt;Conclusions:&lt;/b&gt; The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation

Transition of plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein pumilio

Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism

Zoonotic Diseases Report 2014

De Staat van zoönosen geeft jaarlijks een overzicht van infectieziekten die overgaan van dier op mens, de zogenoemde zoönosen. Het gaat om de mate waarin meldingsplichtige zoönosen voorkomen en de ontwikkelingen daarvan op de lange termijn. Hierbij betreft het zowel het aantal ziektegevallen bij mensen als het voorkomen van deze ziekteverwekkers bij dieren. Ook worden elk jaar opmerkelijke voorvallen uitgelicht en wordt een thema behandeld. Voor de meeste zoönosen zijn in 2014 geen uitgesproken veranderingen waargenomen. Wel is het aantal mensen met leptospirose (waarvan de bekendste vorm de ziekte van Weil is) aanmerkelijk hoger dan het vorige jaar, van gemiddeld 30 gevallen in de afgelopen jaren naar 97 in 2014. Ook steeg het aantal Hantavirusinfecties (van gemiddeld 13 in de voorgaande jaren naar 36 in 2014). UItgelicht Een opmerkelijke gebeurtenis in 2014 is dat twee patiënten in een ziekenhuis zijn opgenomen met een ernstige longontsteking na een infectie met Chlamydia caviae. Beide patiënten bleken thuis cavia's te houden die een luchtweginfectie hadden doorgemaakt. Verder was er sinds 2003 weer een uitbraak van vogelgriep bij pluimveebedrijven veroorzaakt door een hoogpathogeen virus. Hierbij zijn vier van de vijf besmette bedrijven onafhankelijk van elkaar besmet geraakt. Het virus was vermoedelijk afkomstig van trekkende watervogels. Het is onbekend of dit virustype overdraagbaar is op de mens; wereldwijd zijn daar geen gevallen van bekend. Vogels Het thema van dit jaar is 'Onze gevleugelde vrienden' en gaat over zoönosen die via vogels kunnen worden overgebracht, zoals de papegaaienziekte. Hierbij wordt onder andere beschreven op welke vliegroutes van trekvogels gezamenlijke broed- en voederplaatsen liggen waar ze elkaar kunnen treffen en zoönoseverwekkers aan elkaar zouden kunnen overdragen.The Zoonotic Diseases Report provides an annual overview of infectious diseases transmitted from animals to humans, so-called zoonotic diseases or zoonoses. It focuses on the degree to which notifiable zoonoses occur and how they develop over the long term. Specifically, this concerns both the number of human cases and the occurrence of these pathogens in animals. Noteworthy incidents of zoonoses are also highlighted each year and a particular theme is discussed. For most zoonoses, no pronounced changes were observed in 2014. Nonetheless, the number of people with leptospirosis (of which the most well-known form is Weil's disease) was considerably higher than in the previous year, rising from an average of 30 cases in recent years to 97 cases in 2014. The number of Hantavirus infections also rose (from an average of 13 cases in the previous years to 36 cases in 2014). A closer look A notable event in 2014 is the admission of two patients to hospital with serious lung infections after being infected with Chlamydia caviae. Both patients kept guinea pigs at home that suffered from respiratory infections. There was also the first outbreak of highly pathogenic avian influenza since 2003 affecting five poultry farms. Four of the five farms became contaminated independently of each other. It is thought that the virus was transmitted from waterfowls. It is not known whether the particular virus strain can be transmitted to humans; worldwide no cases of such transmission have been reported. Birds This year's theme is 'Our winged friends'. It focuses on zoonoses that can be transmitted by birds, such as psittacosis (parrot fever). The migratory routes that coincide with shared breeding and feeding locations where migratory birds can come into contact with one another and thereby possibly transmit zoonotic pathogens to each other are also described.NVW

Stakeholder narratives on trypanosomiasis, their effect on policy and the scope for One Health

Background This paper explores the framings of trypanosomiasis, a widespread and potentially fatal zoonotic disease transmitted by tsetse flies (Glossina species) affecting both humans and livestock. This is a country case study focusing on the political economy of knowledge in Zambia. It is a pertinent time to examine this issue as human population growth and other factors have led to migration into tsetse-inhabited areas with little historical influence from livestock. Disease transmission in new human-wildlife interfaces such as these is a greater risk, and opinions on the best way to manage this are deeply divided. Methods A qualitative case study method was used to examine the narratives on trypanosomiasis in the Zambian policy context through a series of key informant interviews. Interviewees included key actors from international organisations, research organisations and local activists from a variety of perspectives acknowledging the need to explore the relationships between the human, animal and environmental sectors. Principal Findings Diverse framings are held by key actors looking from, variously, the perspectives of wildlife and environmental protection, agricultural development, poverty alleviation, and veterinary and public health. From these viewpoints, four narratives about trypanosomiasis policy were identified, focused around four different beliefs: that trypanosomiasis is protecting the environment, is causing poverty, is not a major problem, and finally, that it is a Zambian rather than international issue to contend with. Within these narratives there are also conflicting views on the best control methods to use and different reasoning behind the pathways of response. These are based on apparently incompatible priorities of people, land, animals, the economy and the environment. The extent to which a One Health approach has been embraced and the potential usefulness of this as a way of reconciling the aims of these framings and narratives is considered throughout the paper. Conclusions/Significance While there has historically been a lack of One Health working in this context, the complex, interacting factors that impact the disease show the need for cross-sector, interdisciplinary decision making to stop rival narratives leading to competing actions. Additional recommendations include implementing: surveillance to assess under-reporting of disease and consequential under-estimation of disease risk; evidence-based decision making; increased and structurally managed funding across countries; and focus on interactions between disease drivers, disease incidence at the community level, and poverty and equity impacts

Livestock trade networks for guiding animal health surveillance

BACKGROUND: Trade in live animals can contribute to the introduction of exotic diseases, the maintenance and spread endemic diseases. Annually millions of animals are moved across Europe for the purposes of breeding, fattening and slaughter. Data on the number of animals moved were obtained from the Directorate General Sanco (DG Sanco) for 2011. These were converted to livestock units to enable direct comparison across species and their movements were mapped, used to calculate the indegrees and outdegrees of 27 European countries and the density and transitivity of movements within Europe. This provided the opportunity to discuss surveillance of European livestock movement taking into account stopping points en-route. RESULTS: High density and transitivity of movement for registered equines, breeding and fattening cattle, breeding poultry and pigs for breeding, fattening and slaughter indicates that hazards have the potential to spread quickly within these populations. This is of concern to highly connected countries particularly those where imported animals constitute a large proportion of their national livestock populations, and have a high indegree. The transport of poultry (older than 72 hours) and unweaned animals would require more rest breaks than the movement of weaned animals, which may provide more opportunities for disease transmission. Transitivity is greatest for animals transported for breeding purposes with cattle, pigs and poultry having values of over 50%. CONCLUSIONS: This paper demonstrated that some species (pigs and poultry) are traded much more frequently and at a larger scale than species such as goats. Some countries are more vulnerable than others due to importing animals from many countries, having imported animals requiring rest-breaks and importing large proportions of their national herd or flock. Such knowledge about the vulnerability of different livestock systems related to trade movements can be used to inform the design of animal health surveillance systems to facilitate the trade in animals between European member states. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-015-0354-4) contains supplementary material, which is available to authorized users